Download Visualizing expression patterns of Shh and Foxf1 genes

Pediatr Surg Int (2008) 24:3–11
DOI 10.1007/s00383-007-2036-1
ORIGINAL ARTICLE
Visualizing expression patterns of Shh and Foxf1 genes
in the foregut and lung buds by optical projection
tomography
Hideaki Sato Æ Paula Murphy Æ Shay Giles Æ
John Bannigan Æ Hajime Takayasu Æ Prem Puri
Published online: 26 October 2007
Ó Springer-Verlag 2007
Abstract Congenital malformations of the foregut are
common in humans. The respiratory and digestive tubes
are both derived by division of the foregut primordium.
Sonic hedgehog (Shh) and Fork head box F1 (Foxf1)
genes encode regulatory molecules that play a pivotal role
in gut and lung morphogenesis and are therefore important candidate genes to be examined in models of foregut
developmental disruption. Optical projection tomography
(OPT) is a new, rapid and non-invasive technique for
three-dimensional (3D) imaging of small biological tissue
specimens that allows visualization of the tissue distribution of RNA in developing organs while also recording
morphology. To explore the application of OPT in this
context, we visualized Shh and Foxf1 gene expression
patterns in the mouse foregut and lung buds at several
stages of development. Time-mated CBA/Ca mice were
harvested on embryonic days 9–12. The embryos were
stained following whole mount in situ hybridization with
labelled RNA probes to detect Shh and Foxf1 transcripts
at each stage. The embryos were scanned by OPT to
obtain 3D representations of gene expression domains in
the context of the changing morphology of the embryo.
H. Sato H. Takayasu P. Puri (&)
Children’s Research Centre, Our Lady’s Children’s Hospital,
Crumlin, Dublin, Ireland
e-mail: [email protected]
P. Murphy
Department of Zoology, University of Dublin, Trinity College,
Dublin 2, Ireland
S. Giles J. Bannigan
School of Medicine and Medical Science,
University College Dublin, Belfield, Dublin, Ireland
OPT analysis of Shh and Foxf1 expression in the foregut
and lung buds revealed extra details of the patterns not
previously reported, particularly in the case of Foxf1
where gene expression was revealed in a changing pattern
in the mesenchyme around the developing lung. Shh
expression was also revealed in the epithelium of the lung
bud itself. Both genes were detected in complementary
patterns in the developing bronchi as late as E12, showing
successful penetration of molecular probes and imaging at
later stages. OPT is a valuable tool for revealing gene
expression in an anatomical context even in internal tissues like the foregut and lung buds across stages of
development, at least until E12. This provides the possibility of visualizing altered gene expression in an in vivo
context in genetic or teratogenic models of congenital
malformations.
Keywords Oesophageal atresia Foregut Sonic hedgehog Foxf1 Optical projection tomography
Introduction
Congenital malformations of the foregut such as the
oesophageal atresia/tracheo-oesophageal fistula (OA/TOF)
are common in humans. Until recently, little was known
about the pathogenesis of these anomalies and there was
little opportunity to study them. The accidental finding that
the anthracycline antibiotic Adriamycin has teratogenic
effects on rats, producing tracheo-oesophageal malformations has provided a reproducible model [1]. This
Adriamycin rat model has contributed to investigating the
mechanisms of OA/TOF congenital anomalies [2–4]. The
mouse is the foremost mammalian model of development,
offering an expanding wealth of genetic and molecular
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knowledge and scientific research techniques. Our group
has confirmed that the Adriamycin mouse model (AMM)
produces a spectrum of tracheo-oesophageal malformations
[5, 6].
Sonic hedgehog (Shh) is a secreted glycoprotein that
acts as a cell signalling molecule and has multiple patterning roles in the developing embryo. The Shh gene is
expressed in various organizing centers, such as the
notochord, floor plate of the neural tube and polarizing
region of the limbs [7]. It is involved in dorso-ventral
foregut patterning [7, 8], presumably through notochord
localization. The later expression of Shh in lung buds
suggests that it plays a role in the morphogenesis of
various epithelial appendages of the respiratory system [8,
9]. Homozygous Shh mutant mouse embryos are characterized by a number of development defects; especially in
the respiratory tract that include failure of the trachea to
develop as a separate structure from the oesophagus [8,
10]. In Adriamycin treated rats, the level of Shh protein
expression is very low, without any time-dependent
changes [11, 12].
The transcription factor Fork head box F1 (Foxf1) gene
encodes a regulatory molecule that plays a pivotal role in
gut and lung morphogenesis, presumably through the regulation of cell-specific target genes and is therefore an
important candidate gene to be examined in models
of foregut developmental disruption [13, 14]. Foxf1 is
expressed in the foregut mesoderm and its expression is upregulated by Shh [15]. In mice, tracheo-oesophageal morphogenesis is highly sensitive to the dosage of Foxf1.
Heterozygous Foxf1 embryos show variable phenotypes
that include narrowing of the oesophagus and trachea,
oesophageal atresia, tracheo-oesophageal fistula, and lung
hypoplasia [14, 16]. Similar anomalies were also demonstrated in mice lacking in Shh, and it has been suggested
that Foxf1 is involved in the Shh signalling pathway,
downstream of Shh [15].
Recently, various tools for obtaining three-dimensional
(3D) information on biological tissues have been investigated and improved [17–19]. Optical projection tomography
(OPT) is a new, rapid and non-invasive technique for 3D
imaging of small biological specimens that allows visualization of the tissue distribution of RNA, protein or
histological stains in developing organs while also recording
morphology [20]. To explore the application of OPT to the
study of a congenital model, such as AMM, we visualized
Shh and Foxf1 gene expression patterns in the mouse foregut
and lung buds at several stages of development. This enabled
us to visualize not only morphological development, but
underlying molecular changes, such as gene expression.
Here, the strengths and limitations of the approach are
presented.
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Materials and methods
Animals
Male and female CBA/Ca mice (Harlan UK, Bicester,
England) were accurately time-mated over a 4 h period
starting at 8 a.m. Identification of a vaginal plug at the
end of the mating period was taken to be the start of
gestation.
The dams were humanely killed by swift cervical dislocation between embryonic day (E) 9 and E13. The
embryos were washed with phosphate buffered saline
(PBS) and then fixed in 4% paraformaldehyde in Rnasefree PBS overnight. The embryos to be used for in situ
hybrydization were stored at –20°C following dehydration
through a methanol series.
Whole mount in situ hybridization
Before staining, E12 embryos were dissected to include
only the trunk region from the level of the first branchial
arch to the base of the liver. Whole mount in situ hybridization was performed on embryos using digoxygeninlabelled riboprobes for Shh and Foxf1 (generous gift of Dr
L.Lundh). This technique involved embryo pretreatment
followed by overnight hybridization with the probe,
application of the antidigoxygenin antibody (Roche,
Mannheim, Germany) and signal development using the
NBT/BCIP solution (Roche, Mannheim, Germany). Whole
embryos were examined and photographed to view the
externally visible pattern of gene expression.
Optical projection tomography
OPT was carried out as described in Sharpe et al. (20).
Stained embryos were embedded in a 1.0% low melting
point agarose in H2O and attached to a metal mount. The
mounted embryos were then dehydrated through 100%
methanol prior to clearing by immersion in a 2:1 benzyl
alcohol:benzyl benzoate solution. The specimen was then
loaded into a prototype OPT scanner built at the Medical
Research Council Human Genetics Unit and installed in the
Zoology Department, Trinity College, Dublin. A total of
400 projection images were captured using a Leica
MZFLIII stereo-fluorescent microscope through a full 360°
rotation of the specimen. Images were captured either in
visible light (to capture the colorimetric staining of the
gene expression pattern) or in UV light with a Texas Red
filter to capture embryo morphology. The captured data
were transferred to a Linux computer where a series of
Pediatr Surg Int (2008) 24:3–11
programmes (provided by the Edinburgh Mouse Atlas
Project EMAP) performed a back projection algorithm to
reconstruct 3D representations of the specimens. Reconstructed data were viewed and analysed using software
provided by EMAP.
All experiments were carried out in compliance with the
current European Union regulations for animal investigation (ED86/609/EC), with prior ethical approval under
licence from the Department of Health, Ireland.
Results
Analysis of computer reconstructed specimens following
OPT allowed the Shh and Foxf1gene expression patterns to
be viewed in 3D, either externally as ‘‘volume rendered’’
(vr) 3D movies (still shots from these movies are shown in
Figs. 1, 2, 3, 4), or following virtual sectioning to reveal
the patterns of transcript distribution through the developing tissues. The following is a description of the patterns
revealed across stages E9–E12, with emphasis on foregut
and lung bud expression. This shows the advantage of
viewing the full pattern in the context of the changing
morphology of the tissues at each stage and the potential of
revealing possibly altered expression in the context of
altered morphology in a congenital model system.
Shh expression
In E9 embryos (Fig. 1a–e), OPT revealed the expression of
Shh in the floor plate of the neural tube, the notochord and
the foregut where transverse sections (Fig. 1c, d) clearly
show localization of Shh transcripts to the ventral aspect of
the gut invagination, shown here at the level of the pharyngeal arches. The precise domain of the ventral
localization could not be ascertained from whole mount
preparations (Fig. 1a), showing the advantage of 3D
computer representation where the data can be virtually
sectioned and analysed in multiple orientations.
At E10, Shh expression persisted in the floor plate of the
neural tube, notochord, foregut and hindgut, as is visible in
the raw whole mount in situ hybridization image and the
3D computer representation (Fig. 1f, g). In the foregut,
OPT analysis of the specimen clearly showed extra details
of the pattern; restriction to the ventral endoderm of the
laryngotracheal groove and through the emerging lung
buds (Fig. 1h–j), including the site of division of the
respiratory groove from the foregut (Fig. 1g).
At E11, external views of in situ hybridized embryos
showed Shh expression again in the floor plate of the neural
tube, and now also detectable in the polarizing regions of
the fore and hind limb buds (Fig. 1k, l). Expression in the
5
gut was no longer clearly visible externally in the raw
specimen (Fig. 1k) as the size of the embryo increased, but
was clearly visible following OPT scanning (Fig. 1l).
Section analysis of the OPT reconstruction clearly showed
Shh expression through the foregut, the branching division
of the trachea and in the lung buds (Fig. 1n–q), with the
ventral restriction in the endoderm of the laryngotracheal
groove (Fig. 1o) extending into the dorsal territory in more
caudal sections (Fig. 1p), and lung buds (Fig.1q).
At E12, embryos were enlarged considerably and the
complexity of the tissues increased so that successful
whole mount in situ hybridization was challenged due to
reduced penetration of the RNA probe and components of
the detection method. To ensure penetration, the embryos
were dissected to include only the trunk region from the
first branchial arch to the base of the liver. In such preparations, internal expression cannot be viewed easily in raw
specimens, although part of the floor plate and stomach are
visibly stained (Fig. 2a). Following OPT scanning and
reconstruction, details of the expression in the endoderm of
the oesophagus and lung buds could be seen (Fig. 2b–e).
Foxf1 expression
External analysis of whole mount preparations showing
Foxf1 expression in E9 embryos (Fig. 3a) showed strong
staining in the anterior mesenchyme in the region of the
foregut and in the ventral part of the neural tube (Fig. 3a).
The superficial pattern appeared very similar to that of Shh.
OPT analysis clearly revealed the precise distribution of
Foxf1 transcripts in the floor plate of the neural tube and
the notochord, similar in this respect to Shh expression, and
in the mesenchyme associated with the foregut (Fig 3c–e),
adjacent to the expression of Shh in the ventral foregut
endoderm. Foxf1 expression in E10 embryos was again
visible in whole-mount preparations in the ventral CNS,
particularly strongly in the hindbrain, midbrain and diencephalons, and in the viscera at the level of the foregut. It
was also visible in the posterior fore-limb buds (Fig. 3f).
From OPT analysis, the distribution in the viscera became
clear, again in the mesenchyme around the foregut, most
intense in the ventral aspect and around the lung buds
(Fig. 3g–k). In addition, there was particularly strong
expression of Foxf1 in a posterior enlargement of the
foregut where the stomach was developing (Fig. 3k).
Superficial analysis of E11 embryos, hybridized to
reveal Foxf1 expression (Fig. 3l), showed staining again in
the ventral neural tube, but appeared to be restricted to the
brain region and the trunk between the levels of the limb
buds. However, OPT analysis showed that ventral neural
tube expression was continuous (Fig. 3m) and that there
was an apparent discontinuity is an artefact of viewing
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Fig. 1 The expressions of Shh at E9–E11. a–e E9, f–j E10, k–q: E11.
a, f and k Photomicrographs of lateral views of whole mount Shh in
situ hybridized specimens. b, g and l Still shots taken from movies of
the externally visible patterns of 3D computer reconstructions
following optical projection tomography (OPT) scanning (vr views).
Note the enhanced visualization of the expression patterns, especially
of more deeply stained tissue, such as the foregut and lung buds. c, h
and n Virtual sections taken through the computer reconstructions in
coronal planes. m A virtual section in a sagittal plane. d, e, i, j, o, p, q
Transverse virtual sections taken in the planes indicated by lines on
(c), (h) and (n). Arrowheads indicate foregut expression and arrows
indicate respiratory tract expression. The arrowheads in (d) and (e)
highlight the ventral restriction of Shh expression in the foregut. The
arrow in (g) indicates the beginning of division of the respiratory
groove from the foregut. Arrows in (h) and (j) indicate Shh expression
in the lung buds. Arrowheads in (i) indicate ventral expression in the
anterior foregut. m The expression of Shh in the division of trachea
from foregut is visible (arrow). In (o)–(q), the restriction of Shh
expression to ventral endoderm in the laryngotracheal groove
(arrowhead in (o)), extending into the dorsal territory in more caudal
sections (arrowhead in (p)) and lung buds (arrows in (q)) is visible.
Scale bar a = 0.3 mm, f = 0.5 mm, l = 0.8 mm. a anterior, d dorsal,
nt neural tube, nc notochord, h heart, fg foregut, hg hindgut, mb
midbrain, fl forelimb, hl hindlimb, lb lung bud
internal expression through an enlarged embryo, highlighting the need for OPT reconstruction and analysis.
Expression increased in the posterior limb buds. OPT
analysis showed the distribution of transcripts with respect
to the division of trachea from the foregut and in the
mesenchyme surrounding the lung buds (Fig. 3m–s). There
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Fig. 2 Shh expression at E12 in the dissected embryonic trunk from
the first branchial arch to the base of the liver. a Photomicrograph of
the raw specimen. Part of the floor plate and stomach are visibly
stained. b A still from a vr movie showing the externally visible
pattern in the 3D computer reconstruction of the specimen following
OPT, enabling clear visualization of Shh expressions in the floor plate
of the neural tube and in the endoderm of the oesophagus
(arrowhead) and lung bud (arrow). c A virtual midline sagittal
section through (b); Shh expression in the endoderm of the
oesophagus is clearly visible. d A coronal section through (b); Shh
expression in the endoderm of the lung buds is visible. e, f Transverse
sections through (b) in planes indicated on (d). The OPT clearly
visualized Shh expressions in the endoderm of the oesophagus
(arrowhead in (e)) and lung buds (arrowhead in (f)). Scale bar
a = .1.5 mm. a anterior, d dorsal, h heart, fl forelimb
was still strong expression in the forming stomach
(Fig. 3s).
The dissected trunk region of E12 embryos (Fig. 4a), as
in Fig. 2, showed little detail on superficial examination
due to tissue density, but virtual sections showed expression in the splanchnic mesoderm surrounding the lung buds
in a complementary pattern to the expression of Shh in the
lung bud endoderm (compare Fig. 2c–f with Fig. 4c–f).
Foxf1 is also now expressed in the splanchnic mesoderm
around the oesophagus (Fig. 4c, f).
from images of serial sections presents a number of technical problems. First of all, it is extremely labour intensive
and time consuming, and, secondly, loss or distortion of the
sections can cause inaccuracies in the 3D reconstruction.
Another challenge for 3D imaging of embryonic events is
the need to capture fluorescent dyes that are now commonly used to stain for the distribution of molecules in the
embryonic tissues. Confocal microscopy can be used to
image fluorescent stains with very high resolution, but only
in small tissue samples several hundred microns in depth.
In addition, this technique is limited to imaging only
fluorescent staining and is not suitable for analysis of
coloured dyes such as BCIP/NBT detection that are widely
used to stain for the distribution of gene transcripts following whole mount in situ hybridization: a technique that
adds very valuable information about gene activity in the
context of the developing embryo [18]. Magnetic resonance imaging (MRI) can also be used for 3D imaging of
internal morphology without the need for physical sectioning, but cannot capture fluorescent or colorimetric
stains and is also limited in the size range it can feasibly
cope with. Despite improvements to the resolution of MRI
Discussion
The need for 3D visualization of events in the developing
embryo has previously been recognized and addressed in a
number of recent studies [17–22]. Williams et.al. and
Sasaki et.al. [21, 22] described tracheo-oesophageal separation during embryogenesis by compiling sequential 2D
images from H & E stained sections at several stages of
foregut development. They clearly demonstrated the
developmental events in great detail, but reconstruction
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Fig. 3 The expressions of
Foxf1 at E9–E11. a–e E9, f–k
E10, l–s E11. a, f, l
Photomicrographs of lateral
views of whole mount Foxf1 in
situ hybridized specimens. b, g,
m Still shots taken from movies
of the externally visible patterns
of 3D computer reconstructions
following optical projection
tomography (OPT) scanning (vr
views). c, h, o, p Virtual
sections taken through the
computer reconstructions in
coronal planes. n A virtual
section in a sagittal plane. d, e,
i, j, k, q, r, s Transverse virtual
sections taken in the planes
indicated by lines on (c), (h), (o)
and (p). Arrowheads indicate
foregut expression and arrows
indicate respiratory tract
expression. The arrowheads in
(d) and (e) highlight Foxf1
expression in the mesenchyme
of the foregut. Arrows in (j)
indicate Foxf1 expression in the
mesenchyme around the lung
buds. Arrowhead in (k)
indicates Foxf1 expression in
mesenchyme of the foregut
where the stomach is
developing. q–s The expression
of Foxf1 to mesenchyme
surrounding the laryngotracheal
groove (arrowhead in (p)),
extending into the mesenchyme
around lung buds in more
caudal sections (arrows in (s)).
Scale bar a = 0.3 mm,
f = 0.5 mm, l = 0.8 mm. a
anterior, d dorsal, nt neural tube,
nc notochord, h heart, fg
foregut, hg hindgut, mb
midbrain, fl forelimb, hl
hindlimb, lb lung bud
where it is possible to image specimens in the size range of
mammalian embryos, it requires very special magnets that
are extremely expensive and not widely available [19].
OPT is a relatively new imaging technique that is custom
built to visualize specimens in the size range (0.5–10 mm)
of mammalian embryos with high resolution, revealing
details of developing organs within [20]. The technique is
non-invasive (does not require sectioning), avoiding the
problem of section loss and distortion, allows capture of
coloured or fluorescent stained tissue to show gene
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expression patterns and the distribution of other molecules
within the context of the rapidly changing morphology of
the developing embryo. OPT fills an important imaging gap
between confocal microscopy and MRI, and gives important information on the distribution of developmentally
active molecules. In this study, we show the use of OPT to
visualize the distribution of the transcripts of two genes,
Shh and Foxf1, which are prime candidates for involvement in the congenital malformations associated with OA/
TOF, across stages of development when malformations
Pediatr Surg Int (2008) 24:3–11
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Fig. 4 Foxf1 expression at E12 in the dissected embryonic trunk
from the first branchial arch to the base of the liver. a Photomicrograph of the raw specimen. Part of the floor plate and lung are visibly
stained. b A still from a vr movie showing the externally visible
pattern in the 3D computer reconstruction of the specimen following
OPT, enabling clear visualization of Foxf1 expressions in the floor
plate of the neural tube and in the mesenchyme of the lung. c A virtual
midline sagittal section through B; Foxf1 expression in splanchnic
mesoderm around the oesophagus is clearly visible. d A coronal
section through (b); Foxf1 expression in splanchnic mesoderm around
the lung buds is visible. e, f Transverse sections through (b) in planes
indicated on (d); the OPT clearly visualized Foxf1 expressions in the
splanchnic mesoderm around the lung buds (arrowhead in (e), (f))
and oesophagus (arrow in (f)). Scale bar a = .1.5 mm. a anterior,
d dorsal, h heart, fl forelimb
occur in the mouse model AMM. This illustrates the value
of our current approach in analysing the molecular and
morphological alterations that occur in this mouse model.
This approach depends on the coupled techniques of
whole mount in situ hybridization (WISH) and OPT
imaging. One limitation is the difficulty of extending the
study to later stages of development, due to reduced penetration of the molecular probes and staining components
involved in WISH. Here, we show successful staining and
visualization of deep staining by combining with OPT
scanning up to E12. It is as yet unknown how much more
we can extend the method by further dissection of the
tissue. This is currently under investigation.
In normal development, the respiratory and digestive
tubes are derived by division of the foregut primordium
that is surrounded by splanchnic mesoderm [23]. In mice,
the bronchi are the first respiratory structures to be generated as bulges on the ventrolateral wall of the foregut at E
9.5. At E10, the laryngo-tracheal groove gives rise to the
trachea, which connects to the left and right main bronchi.
The lung buds are located on the ventral aspect of the
gastric dilatation of the foregut. The tracheal and bronchial
stalks extend to form the main bronchi, a process completed by approximately E10.5. Stereotypic branching and
budding occurs as the bronchial and bronchior tubules form
between approximately E11 and E16 [24]. But the
embryological development of the foregut into a respiratory and a digestive part is a complicated process, of which
many aspects have not yet been clarified, particularly the
factors that trigger the normal progression of events
described above. In OA/TOF conditions, it is thought that a
combination of genetic and environmental factors plays a
role in the aetiology of foregut anomalies. Some morphological changes, such as branching of the notochord, have
been noted in the Adriamycin treated model [25]. The
notochord therefore may play a role in guiding normal
development of the foregut in this respect. It will therefore
be of great interest to visualize any alterations to the
morphological progression of events as well as of expression patterns of prime candidate genes in the mouse model.
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Shh plays multiple crucial roles as a signalling molecule
during development in both invertebrate and vertebrate
species. The localized expression of Shh plays a role in the
morphogenesis of various epithelial structures such as hair,
teeth, the respiratory system and the gut [26]. Specifically,
in the respiratory system, Shh expression in the respiratory
primordium from E9.5 in the mouse has an important role
in lung branching [27]. In the foregut, the dorso-ventral
restriction of Shh expression from E10 to E12 has previously been reported by several researchers [8, 28, 30], and
mice in which the Shh gene has been inactivated display
abnormal foregut morphogenesis resulting in oesophageal
atresia, failed separation of the trachea and oesophagus,
and lungs that arise from a single tracheo-oesophageal tube
[8]. This indicates the important role of Shh in normal gut
and respiratory tract development and the importance of
Shh as a candidate gene for involvement in OA/TOF
malformations. Ioannides et al. investigated the involvement of Shh in malformations of the trachea and
oesophagus in the AMM. They revealed a lack of dorsoventral patterning in the foregut of the AMM, and suggest
that the disturbance of this pattern may underline abnormal
organogenesis [30]. Williams et.al. [21] showed the morphological changes in the notochord in the AMM and
suggested that the proximity of the notochord to the foregut
or its associated mesenchyme may be key to the induction
of abnormalities. Arsic et al. [29] further suggested that a
possible consequence of the abnormal proximity of the
notochord to the foregut in Adriamycin treated embryos is
that the foregut expression of Shh may be repressed by
signals from the notochord. Shh is therefore a prime and
topical candidate to be examined in this model. Our preliminary work presented here captures the complete 3D
distribution of Shh transcripts during normal development,
showing important time-dependent spatial changes in the
pattern. In particular, it captured the details of dorso-ventral restriction in the foregut and enabled us to visualize the
expression of Shh in the developing respiratory system,
especially as the primordia separate from the foregut. This
is a framework for the comparison of Adriamycin and
control treated embryos in an ongoing work.
Foxf1, previously known as Hfh8 or FREAC1, is a
member of the forkhead family that is involved in foregut
development. Foxf1-null mutant mice die in utero before
embryonic day 10, due to extra-embryonic mesoderm
defects. A study by Mahlapuu et al. [15] showed that heterozygous mice have a high perinatal mortality and exhibit
multiple defects in foregut-derived structures, including
lung hypoplasia, lung immaturity and lobulation defects as
well as OA/TOF. Similar anomalies were also demonstrated
in mice lacking the Shh gene and it has been suggested that
Foxf1 acts downstream in the Shh pathway [15]. Hence,
Foxf1 plays an important role in the development of the
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foregut. The expression of Foxf1 in the developing embryo
is less comprehensively described in literature then that of
Shh. Expression of Foxf1 has been reported in the
splanchnic mesoderm surrounding the gut endoderm at
E9.5, in the lung diverticulum, developing intestine and
allantois at E10.5, and in the developing lung, stomach,
intestine and intersomitic arteries at E12 [16]. In our study,
OPT analysis enabled us to not only represent the patterns in
3D, but to add significant details to the known expression
patterns. In the lung bud comparison with Shh expression
(compare Figs. 1 and 3 and 2 and 4) highlighted the complementarity of the two patterns, supporting the idea of
Foxf1 responding downstream of Shh signalling. In the
developing digestive tract, particularly strong expression in
the developing stomach was noted for the first time.
These results support the use of WISH coupled with
OPT imaging as a valuable tool for revealing gene
expression in an anatomical context, even in internal tissues like the foregut and lung buds across stages of
development, at least until E12. This provides the possibility of visualizing altered gene expression in an in vivo
context in genetic or teratogenic models of congenital
malformations.
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